Binding assays are routinely used to screen for and diagnose a host of diseases and conditions, including Lyme disease, herpes, acquired immunodeficiency syndrome (AIDS), streptococcal infections, lupus and pregnancy. Such assays are relatively simple in theory, utilizing the binding affinity between two or more binding members to detect and/or quantify the presence of one of the members, referred to herein as the analyte. Binding members comprise a wide range of substances, including antigens, antibodies, haptens, complimentary nucleic acid sequences, ligands, small molecules and receptors. Antigen-antibody binding member pairs used in immunoassays currently enjoy the most widespread use.
A common format of a binding assay involves immobilizing a binding member specific for the analyte on a paper-like sheet or membrane. The membrane is then contacted with the test sample and appropriate reagents under conditions allowing binding to occur between the immobilized binding member and any analyte in the sample, with means for detecting binding events also provided. Often a labeled second binding member which binds to the first binding member-analyte complex is added to provide a detectable signal on the membrane.
The sandwich immunoassay is an example of one commonly used binding assay for antibody detection. In a generic sandwich immunoassay, the antigen is immobilized on a solid substrate. Antibody containing solution, e.g., diluted serum, is incubated with the immobilized antigen. Antibodies specific to the antigen bind to it, and unbound antibodies are then removed by buffer washes. A detection agent which may typically be a secondary antibody conjugated to an enzyme, is then incubated with the primary antibody-antigen complex. Finally, an enzyme substrate is added which is converted into a visual, detectable product whenever the enzyme is. Such multi-step sandwich immunoassays can be developed in many different ways depending on assay requirements.
A Western Blot is another example of a commonly used immunoassay. In current practice, the Western Blot method comprises a sequence of incubation and wash steps performed on a membrane bearing electrophoretically resolved antigen bands. Typically, the membrane is cut into narrow strips, each bearing the identical pattern of antigen bands. Strips are then processed in reagent solutions individually in narrow trays, each typically holding 0.5-2.0 ml. In the first step, the strip is incubated with a blocking solution containing a non-specific protein, e.g., non-fat dry milk, bovine serum albumin, newborn calf serum or gelatin. After washing off excess blocking solution with a wash buffer, typically a physiological saline buffer containing a low percentage of detergent, the strip is then incubated with antibody solution. Antibody solution may be diluted human or animal serum, cerebrospinal fluid, dried blood spot eluate, monoclonal antibody, to name a few. Unbound antibody is then washed off with buffer, and the strip is incubated in the detection reagent. In a typical application, the detection reagent could be goat-anti-human IgG-alkaline phosphatase conjugate. Unbound detection reagent is washed off with buffer, and finally the substrate (for alkaline phosphatase, a common substrate is 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium) for the detection enzyme is added. The conversion of the substrate to a visually detectable product is allowed to proceed until optimal visualization of bands, and then substrate is washed away. The strip is typically dried, providing a permanent record of the assay result. Bands on the strip indicating antibody reactivity can be compared with control strips to determine the specificity of the immunoreaction. In currently used algorithms for HIV and Lyme testing, a positive test result is defined as the appearance of certain combinations of specific bands. For example, an HIV Western Blot test requires the presence of two bands to be considered positive, while a Lyme Western Blot test requires five out of ten bands to be positive for IgG, or two out of three bands to be positive for IgM.
As described above, the Western Blot method involves incubating the membrane strips sequentially in reagent solutions usually contained in a tray. In typical protocols, incubations with antibody solutions and detection reagents may take 30 minutes to several hours each. Wash steps may take 5-10 minutes each. The total time for processing a blot is therefore, not less than one hour, and is often several hours.
It would thus be desirable to provide a method and apparatus for the rapid processing of a binding assay, e.g., an immunoassay, including a Western Blot.